Vaccines to Prevent Cholera

MYRON M. LEVINE AND WILBUR H. CHEN VACCINOLOGY IN LATIN AMERICA enterotoxin maycausemore severe clinicaldiseasethanbonafideEl Tor strains. to enterocytes asakey stepinthepathogenesis of cholera. enterotoxin andsometimesclassicaltoxin co-regulated theorganelles pili(TCP), bywhich of theElTor biotype.Emerging ElTor variantshave beenidentified that express classicalbiotypecholera two biotypes,classicalorElTor. Almostallcholeradiseasecurrently occurringintheworldisduetovariants serotypes, InabaandOgawa;athird serotype, Hikojima, israre. Serogroup O1strainsare alsoclassifiedinto enterotoxin andattachment piliandcauseepidemiccholera. Circa 206 Oserogroups of Etiologic Agents such epidemicsaccompanycomplexemergencies (earthquakes, floods,wars). following vaccination) that mayallowittobeamenablethecontrol of virgin soilepidemics, of asingledose.Oneof thenewvaccineshascharacteristics(singledose,protection asearly8–10days confer ahighlevelof protection uponimmunologically-naïve personswithinjustafew daysof administration public healthauthoritiesanddisruptcivilsociety. Thecontrol of suchepidemicsdemandsavaccinethat can followed adevastating earthquake. when cholerareturned toSouthAmericain1991after acenturyof absenceandthe2010outbreak inHaitithat large explosiveepidemicsinimmunologically-naïvepopulations (socalled“virgin soil”epidemics)suchas or endemic.Theremaining potentialuseof choleravaccines,arguably themostimportant,aimstocontrol travelers from cholera-free countries/regions whomusttravel tocountries/regions where choleraisepidemic populations suchasrefugees internedincampscholera-endemicorcholera-proximal areas, andprotect in recent yearsare employedtodampentheintensityof seasonaldiseaseinendemicareas, protect highrisk pandemics involvingmanycountriesoveryears.Theoralcholeravaccinesthat have becomeavailable gravis”, leadingtodeath ifuntreated), itsexplosiveepidemicbehavior anditspropensity tooccurin extensive of cardinal publichealthimportancebecauseof theseverityof theclinicalillnessitcancause(“cholera Cholera, theacutediarrhealdiseasecausedby Introduction University of MarylandSchoolof Medicine,Baltimore, MD Chief, AdultClinicalStudies Section,Centerfor Vaccine DevelopmentandAssociate Professor of Medicine, Wilbur H. Chen,M.D., M.S. University of MarylandSchoolof Medicine,Baltimore, MD Simon andBessieGrollman DistinguishedProfessor; Associate Deanfor GlobalHealth,Vaccinology andInfectious Diseases, Myron M.Levine,M.D., D.T.P.H. Vaccines toPreventCholera 2

Vaccines toPrevent Cholera V. cholerae 1,2 Virgin soilepidemicsseverely straintheresources of national andlocal are recognized butonlytwo,O1andO139, routinely express cholera Vibrio cholerae 5-8 4 These“El Tor hybrids”expressing classical WithintheO1serogroup there are twomain serogroups O1andoccasionallyO139, is 9

V. cholerae 3 particularlywhen attaches Levine and Chen 3

Epidemiology

The Ganges River delta in South Asia is the ancestral home of cholera where between pandemics it persists as “Asiatic cholera.” The seventh pandemic of cholera, due to V. cholerae O1 El Tor, originated in the early 1960s on the island of Sulawesi, Indonesia, and progressively spread in waves over the ensuing six decades to involve at one time or another almost all of the world’s developing and transitional countries;4 in many it has remained endemic in sub-populations and niches.4 Thus, cholera is now endemic in many countries of South and Southeast Asia, sub-Saharan Africa and a few countries in the Americas. During the early 1990s it was endemic for several years in Peru, Ecuador, and some other Latin American countries.10,11

When cholera invades new territory with immunologically-naive populations, the highest incidence of disease is observed in young adult males. If the disease becomes endemic, the incidence increases in women and children and eventually peak incidence is observed in young children. Cholera exhibits a seasonal pattern almost everywhere that it is endemic.12 When the new season begins, cholera cases emerge simultaneously in multiple geographically separate foci. This pattern has also been observed when cholera invades new territory. In 1991, when cholera re-invaded South America with an explosive and extensive epidemic in Peru, large outbreaks appeared almost simultaneously in three distinct cities spanning a 900-kilometer stretch of the Pacific Coast.12 The explosive increase of cases observed at the onset of many epidemics may be the consequence of hyperinfective vibrios released into water sources lacking vibriophages. Conversely, curtailment of the epidemic may be the consequence of an increased prevalence of lytic phages in the water.13,14

Reservoirs of Infection. Humans are the sole known natural host of V. cholerae O1 cholera disease and chronic carriers are rare.15,16 Thus, it was previously assumed that in endemic areas mild and asymptomatic infections served as the reservoir to maintain the disease until the next cholera season when conditions would once again favor enhanced transmission. However, epidemiologic observations in the 1970s refuted this assumption and ushered in a new understanding of cholera epidemiology that clarified much of the epidemiologic behavior that previously had been puzzling. Confirmation of a single case of cholera in Texas in 1973 in a fisherman caused by an unusual highly hemolytic El Tor Inaba strain,17 followed 5 years later by an outbreak of approximately two dozen cases of the identical strain in which poorly cooked seafood was incriminated as the vehicle,18 led to identification of an environmental focus of infection along the Gulf of Mexico coast of the U.S.A.19 This El Tor Inaba strain was found to constitute autochthonous flora of the brackish waters of Gulf estuaries, where it was associated with crustacea (shrimp, etc.) eaten as local seafood. Identification of a similar environmental focus of free-living enterotoxigenicV. cholerae O1 El Tor in Queensland, Australia, supports the hypothesis that brackish water environmental niches can serve as the reservoir of V. cholerae O1.20

V. cholerae can enter a “viable but nonculturable” state that allows them to survive harsh environmental conditions through a form of bacterial hibernation.16,21 When the toxigenic V. cholerae eventually encounter favorable conditions of temperature, salinity and pH, they can rejuvenate, regaining the potential to actively metabolize and grow.21 These may also be the conditions under which zooplankton blooms occur.

Modes of Transmission. Our practical knowledge of the vehicles of transmission of cholera stems from case- control investigations that have documented waterborne transmission and an array of food vehicles.22,23 When El Tor cholera struck the Pacific coast of several Andean countries of South America in 1991, improperly functioning municipal water supplies and sewage systems, contaminated surface waters, and unsafe domestic water storage methods fostered facile waterborne cholera transmission.1,24 Beverages prepared with contaminated water and sold by street vendors, ice, and even commercial bottled water have been incriminated.25 VACCINOLOGY IN LATIN AMERICA purging ricewater stools,toxigenic It hasbeenhypothesized that for afew hoursafter beingshedinenormousnumbersbycholera patients is essentiallyalwaysviafood orwater vehicles. Epidemiologists recognize that person-to-person contactspread of choleravirtuallyneveroccurs.Transmission prior toenteringanotherportthousandsof milesaway. distances canoccurviatheballastwater of large ships,astheyintake ballastwater inoneportand discharge it Finally, ifpathogenic on localcustoms,climate, andotherfactors,particularmodesvehiclesof transmissionpredominate. During outbreaks orseasonalepidemics,choleramayspread viamultiplemodesof transmission.Depending sewage-contaminated water. has alsobeentransmittedbyvegetablesandfruitirrigated withrawsewageor“freshened” bydousingwith these typesof food andstored withoutrefrigeration canincrease byseverallogswithin8to12hours.Cholera Africa. Asmallinoculumof enterotoxigenic Cooked grains,riceandbeanswithsauceshave alsobeenincriminated incholeratransmission,particularly ceviche (uncooked fishorshellfishmarinated inlemonorlimejuice). been raworundercooked seafood, includingmussels,shrimps,oysters,clams,cockles,fish,saltand contaminated duringpreparation orhandling. exoskeletons of shrimp,crabs,andoystersincertainestuarineenvironments, V. cholerae 4 were carriedoutinwhichhundreds of confirmed cases were documented. that Bangladesh hadlittleornocholera.Yet at thesametimelarge-scale fieldtrialstoevaluate choleravaccines WHO ordonotreport at all.Forexample,international health statistics inthelate 1980sand1990sindicated trade, fear of food embargoes andeffects ontourism,manycountriesdelay reporting cholera casestothe the annualburden isestimated tobe1.4–4.0 millioncasesand21,000-143,000 deaths. Forreasons involving were from Africaand15%from theAmericas.Thetruenumberof choleracasesgloballyismuchhigherand 2014, 190,549 casesof cholerawere reported totheWorld HealthOrganization (WHO)from 42countries;55% of diseases.Byinternational convention,choleraisanotifiablediseasealong withplagueandyellow fever. In health surveillanceandreporting wasfirstorganized, itbearsthecode001ininternational classification andDiseaseNotification. International Surveillance is womenof childbearingagewhoexhibitaninordinately highincidence. titer of serumvibriocidalantibodyrise,asincreasing immunityisacquired. often children 1to4yearsof age.Theage-specificincidencefallsthereafter andtheprevalence andgeometricmean chronic gastritis,etc.,have frequently beentheindex case. an immunologicallynaivepopulation, personswithhypochlorhydriafrom partialgastrectomy, risk of developingcholeragravis thanpersonsof otherbloodgroups. Whencholerainvadesanewterritorywith group, Host RiskFactors. transmission exist,theinfectious dosemaybeunusuallylowandspread of diseasemaybeexplosive. of severe choleraoccursinacrowded settingwhere othersusceptiblehumanhostsandfacilemodesof

Vaccines toPrevent Cholera 32,33 hypochlorhydria, O1maybeassociated withseafood vehiclesbymeansof theirnatural adherence tothechitinous Certainhostfactorsmarkedly increase theriskof developingcholera gravis, includingOblood V. cholerae 34,35 andalackof background immunity. 28 O1andO139persistinenvironmental reservoirs, thentransmissionacross long V. cholerae V. cholerae 27 remain inahyper-transmissible state. Themostcommonlyimplicated food vehicleworldwidehas O1introduced byaninfected food handlerintooneof 30 Sincecholerawasthediseasefor whichmodernpublic 37 The highest incidence of cholera in endemic areas is Thehighestincidenceof cholerainendemicareas is 36 Personsof bloodgroup Oare at increased 12 38 One interesting exception to this pattern Oneinteresting exception tothispattern 39

18,21,26 orfood maybesecondarily 13,14,31 Thus,ifacase Helicobacter pylori 13

29

Levine and Chen 5

The Disease

Cholera infection exhibits a spectrum of clinical illness ranging from asymptomatic shedding of vibrios in the stool to life-threatening watery diarrhea accompanied by overt severe dehydration (cholera gravis). Up to three-quarters of cholera infections may be sub-clinical, and among symptomatic patients only a minority may manifest severe purging. The propensity to develop cholera gravis is strongly associated with two host risk factors: blood group O and hypochlorhydria. If the prodigious losses of body water and electrolytes are not promptly replaced in cholera patients who are actively purging “rice water stools” (e.g., at the rate of one liter per hour in an adult), the patient may rapidly dehydrate, suffer renal shutdown, shock and acidosis, and die within hours of the onset of illness. Patients with cholera gravis exhibit the classic signs and symptoms of severe dehydration including weak or absent peripheral pulses, hypotension, sunken eyes, loss of skin turgor, and decreased urine output. Table 1 compares the concentrations of serum electrolytes in normal adult serum and in rice water stools of adults with cholera gravis. The purging of large volumes of rice water stools as evident in cholera gravis is physiologically equivalent to loss of plasma leading to hemoconcentration, hypovolemia, hypotension, decreased renal blood flow, and overt hypovolemic shock.

Table 1. Concentrations of Electrolytes in Normal Adult Sera and in the Rice Water Stools of Adults with Cholera Gravis

Normal Adult Serum Rice Water Stools From Cholera Gravis Patients Na+ 135-145 mEq/ml 135 mEq/ml

K+ 135-145 mEq/ml 15 mEq/ml Cl- 95-105 mEq/ml 100 mEq/ml

HCO3 24-30 mEq/ml 40 mEq/ml

Pathogenesis and Immunity

V. cholerae O1 comprises a sophisticated, multi-step, delivery system for cholera toxin, the virulence attribute responsible for the severe purging of voluminous watery stools characteristic of cholera gravis. In volunteers the ingestion of as little 5 mcg of purified cholera enterotoxin can induce diarrheal illness and 5 mcg has led to a clinical syndrome that closely resembles the severe purging of cholera gravis.40 Subsequent volunteer studies with V. cholerae O1 vaccine strains that harbored deletions in genes encoding the enzymatically active (A) subunit, both A and B (binding) subunits of cholera toxin or the entire cholera toxin virulence cassette (which encodes two other toxins and a minor colonization factor) showed that some strains retained the ability to cause mild diarrhea and other gastrointestinal symptoms,41 possibly by invoking intestinal inflammation.42,43 Whereas ingestion of purified cholera toxin alone can induce a syndrome of severe purging, the fully pathogenic vibrios that cause natural cholera encode multiple virulence factors that direct a stepwise progression to severe diarrhea.

Following ingestion, pathogenic V. cholerae O1 or O139 must survive the formidable gastric acid barrier and transit the pylorus to reach the proximal small intestine, the critical site of host-parasite interaction. Ingestion without buffer of 106 viable pathogenic V. cholerae by fasting North American volunteers resulted in neither infection nor diarrhea because the vibrios were destroyed by gastric acid.44 In contrast, when 106 vibrios are administered with sodium bicarbonate buffer or food that protects the vibrios during gastric transit, cholera VACCINOLOGY IN LATIN AMERICA subsequent experimentalchallengewitheitherthehomologousorheterologous serotpye of serotype (InabaorOgawa)withinabiotypestimulated 90-100%protection againstclinicalillnessupon Experimental challengestudiesinvolunteersshowedthat asingleepisodeof clinicalcholeraduetoeither was unabletocolonize theintestineof volunteersortostimulate goodvibriocidalantibodyresponses. are found withina40-Kb genetically andantigenicallyidenticalbutdiffer somewhat from TCP of classicalbiotype.Genes for TCP biogenesis TCP constitutesthemajorintestinalcolonization factorfor unipolar flagellumpropels theorganisms toward theenterocyte surface, attracted bychemotactins. vibrio enzymesbreak downthemucusbarrieronsurfaceof theintestine,motilityplaysacriticalrole asthe other genesinvolvedwithbacterialadaptation tosurvivalinthehumanintestine.Asneuraminidaseandother thekeypili (TCP), intestinalcolonization factor, of amasterregulatory gene, Once inthesmallintestine,vibriossensetheirenvironment bymeansof ToxR, aprotein that istheproduct who ingesthigherdosesof vibrios. cholerae develops inapproximately 90%of thevolunteers. 6 yet beenidentified. following O139infection are weakandrather nonspecific. Whereas hightitersof specificvibriocidal antibodiesappear after in pediatric cholerapatients, whileserumantibodyresponses toLPSandTCP are more prominent inadults. be aproxy for the stimulation of intestinalantibodies.Serumanti-B subunitresponses are more prominent curiously, serumvibriocidalantibodiesare thebestcorrelate of protection. Whereas infection-derived immunitytocholeraisbelievedbemediated byintestinalmucosalSIgAantibodies, antibody toLPSandCTantigensisagoodmeasure of primingof theintestinal immunesystem. nonprimed individuals.Thedetectionof gut-derived,trafficking IgAantibody secreting cellsthat make specific following cholerainfection. However, significantrisesinSIgAanti-LPS andantitoxin are surprisinglysparsein immunologically primedindividuals,strong secretory IgA(SIgA)intestinalantibodyresponses are recorded are directed toward theOantigenwithremaining 10%of antibodies directed againstprotein antigens.In (IgG) choleraantitoxin are observed. Following Immune Response suggestions that aninitialepisodeof choleraelicitedlittleoronlyshort-livedprotection. potent infection-derived immunitywere corroborated inthefieldwithnatural choleraillness, the protection elicitedbyclassicalbiotypeinfection endured for at leastthree years.

Vaccines toPrevent Cholera O1ElTor causediarrheain~67%of volunteers, V. cholerae O1infection, robust serumvibriocidalantibodyresponses andrisesinimmunoglobulinG Vibrio toxR Pathogenicity Island (VPI). Amutantstrainof Pathogenicity Island(VPI). . 45 Activation of 53,54 Approximately 90% of complement-dependent vibriocidalantibodies 46,47 toxR 44 andtotheindirect activation (via Indeed,whenadministered withbuffer, as few as10 leadstoexpression of choleratoxin andtoxin coregulated 44 V. cholerae althoughthestoolvolumeislessthaninsubjects 59 Acorrelate of protection for O139cholerahasnot V. cholerae O1andO139. V. cholerae 36,56,57 O1infection, vibriocidalresponses Theseserumantibodiesmay 46,47 44,49,50 TCP of ElTor andO139are toxT O1,unabletoexpress TCP, 52 ) of approximately 17 Theseobservations of

12,51 V. cholerae 55 refuting early 48

3

V. V. O1and 58

Levine and Chen 7

Diagnosis

The diagnosis of cholera is confirmed by isolatingVibrio cholerae from stool cultures on selective media such as thiosulfate-citrate-bilesalt-sucrose (TCBS) both directly and after enrichment in alkaline peptone water;60 suspicious colonies are agglutinated with typing sera (directly or after sub-culture). Rapid non-culture tests that detect V. cholerae O1 and/or O139 lipopolysaccharide antigens are useful in field situations.61-63

Treatment

Appropriate antimicrobials are an important adjunct to fluid therapy, as they diminish the volume and duration of purging and rapidly curtail the excretion of vibrios, thereby diminishing the chance of secondary transmission. Patients surviving from hypovolemic shock and severe dehydration manifest certain complications, such as hypoglycemia, that must be recognized and promptly treated. If these fundamental guidelines are followed properly, case fatality, even during explosive epidemics in developing countries, can be kept below 1%.64,65 Failure to comply with these basic proven clinical rules can result in unacceptably high case fatality.66,67

Fluid Therapy. Patients suffering from severe dehydration of cholera with or without overt shock usually lose ~10% of their body weight and must be rapidly rehydrated with intravenous fluids. Fluid therapy is divided into two phases: (1) rehydration phase — the rapid replacement of water and electrolyte deficits, and (2) maintenance phase — the infusion of fluids to replace ongoing losses. Fluid and electrolyte deficits should be replenished as rapidly as possible (within 2–4 hours of initiation). The time recommended for rehydration in adult and pediatric patients is 3 and 6 hours, respectively. In adults, 30% of the total required fluid is administered in the first 30 minutes, while in children this volume is administered over one hour. Patients with cholera gravis generally require multiple liters of intravenous fluids to stabilize them to the point where oral rehydration can begin; at the earliest opportunity, they are carefully weaned from intravenous fluids. Adults with cholera gravis typically require 8–12 liters of intravenous fluids before oral hydration alone can keep up with losses. The most extensively used intravenous rehydration fluid worldwide for treatment of cholera is Ringer’s lactate, because it is so widely available. Ringer’s lactate contains Na+ 130 mEq/L, K+ 4 mEq/L, Ca++ 3 mEq/L, Cl- - + 111 mEq/L, and lactate (precursor of HCO3 ) 29 mEq/L. Because the concentration of K in Ringer’s lactate is too low, supplemental K+ must be administered either by adding a sterile KCl (or similar potassium salt) solution to the Ringer’s solution to increase the concentration of K+ to 15–20 mEq/L, or by initiating oral rehydration.

The volume of all diarrheal losses and vomitus must be measured in the patient with cholera. Once the patient has had replacement of his or her deficit and is in the stage of maintenance therapy, fluid management is generally based on 6-hour periods. The total fluid loss during the previous 6-hour period constitutes the volume of fluids that will be administered to the patient during the next 4-6 hours. As diarrheal losses begin to diminish, the 6-hourly replacement requirements decrease accordingly.

Aggressive rehydration therapy with fluid and electrolytes leads to rapid clinical improvement in the patient (e.g., elevation of blood pressure, stronger pulse, improved skin turgor, and enhanced consciousness) reflected in simple laboratory assays (e.g., fall in hematocrit and plasma specific gravity). Once renal perfusion is re-established normal homeostatic mechanisms begin to combat acidosis and regulate serum electrolyte concentrations. VACCINOLOGY IN LATIN AMERICA may benecessaryinresource constrainedsettings, antibiotic therapy(suchas1 gof ciprofloxacin or300mg of doxycycline for adultsor1gm of azithromycin) is typicallyresistant tothisantimicrobial. sulfamethoxazole useshouldbeavoided inareas where O139isknowntobeprevalent, since dose 1g)wasaseffective asthree days of erythromycin therapy(12.5mg/kg every6hours). children. Inonerandomized, controlled clinicaltrial,asingledoseof azithromycin (20mg/kg, maximum Single-dose azithromycin (1ginadults)hasbeenshowntobeeffective intreating cholerainbothadults and concern withsingle-dosetherapy isthat thismayaccelerate theemergence of resistance. preferred regimen; In areas where tetracycline-resistant antibiotic. cholera precludes stainingof teethand otheradversereactions otherwiseencountered withlongcoursesof this mg/kg for children, for 3to5days).Theveryshortcourse of tetracyclinetherapyused for thetreatment of 3 to5days.Doxycycline requires only oncedailyadministration (300mgfor adultsandteenagers4to6 times dailyfor 3to5daysandthepediatric dosagefor tetracyclineis50mg/kg/day infour divideddosesfor monitoring of vibriostrainsdocumentstheirsensitivity. Theregimen for teenagersandadultsis500mgfour drugs inendemicareas inAsiaandAfricahasdecreased theirutility. Nevertheless,theyremain usefulwhere its longactingderivative, doxycycline, were usedextensivelyinthepasttotreat cholerabutresistance tothese rehydration therapy. Resistanceof stool volume,andduration of excretion of Antimicrobial Therapy. limit isapproximately 750mL/hour. volume of oralrehydration solutionthat canbeconsumedonanhourlybasis;inadultsandteenagerstheupper the basisof a1:1ratio of oralrehydration solutiontovolumeof diarrheal stool.There isapracticallimittothe the Na watery diarrhealstoolpassedinorder toadequately replace Na and-a-half volumesof oralrehydration solutioncontaining90mEq/Lshouldbegivenfor everyvolumeof losses differs byage.SincetheNa The regimen for calculating theamountof oralrehydration solutiontobeadministered toreplace ongoing ORS, somepatients develophyponatremia (albeitusually asymptomatic). controversial for treatment of cholera. rehydration solutions(Na treat cholera; cereal-based oralrehydration solutionsthat provide multipleactivelytransportedsubstrates are usedto packet contains3.5 gof NaCl,2.9 gof sodiumcitrate, 1.5gof KCl, and20gof glucose.InsomeAsiancountries salts andglucosetoprepare 1literof rehydration solutionare widelyavailable indevelopingcountries.Each mEq/L, Cl The oralrehydration solution(ORS)recommended byWHOfor treatment of choleraiscomposedof Na oral rehydration fluidsmustbeingestedto keep upwithongoinglosses. intact duringcholerainfection despitetheeffect of choleratoxin. mediated cotransportof sodiumandwater across themucosalsurfaceof thesmallintestineepitheliumremains managed withoralrehydration alone.Oralrehydration therapyisbasedonthephysiologicalfactthat glucose- Patients withmildormoderate dehydration andmoderate purge rates (<500mLperhour)cangenerallybe 8

Vaccines toPrevent Cholera + concentration of cholerastoolsisonlyapproximately 100mEq/L,ongoinglossescanbereplaced on - 80mEq/L,K 69 somecontrolled trialsshowednoadvantageoverglucose-basedORS. 72 some,butnotall,trialswithsingle-doseciprofloxacin have alsogivengood results. + 20mEq/L,citrate Appropriate antibioticssignificantlydecrease theduration of diarrhea, totaldiarrheal + 75mEq/L,Cl + V.cholerae concentration incholerastoolsisapproximately 135mEq/Linadults,one- V. cholerae 71 - Althoughtherate andvolumeof purging are reduced versusstandard 65mEq/L,K 77 Duringepidemicsindeveloping countries,single-dayorsingle-dose - 30mEq/L,andglucose111mmol/L.Packets containingsufficient V. cholerae O1tocommonlyusedantibioticsisincreasing. Tetracycline and are prevalent ciprofloxacin 250mgoncedaily for 3daysisthe 75,78 + 20mEq/L,citrate , andtherefore constituteanimportantadjunctto particularlyifantibioticsare inshort supply. However, the + losses.Incontrast,inyoungchildren inwhom 68 Ifthediarrheaiscopious,large volumesof - 30mEq/Landglucose75mmol/L)are 70 Reducedosmolarity 76 Trimethoprim– V. cholerae 73-75 + 90 O139

Levine and Chen 9

Cholera Vaccines

There are currently four licensed cholera vaccines, all administered orally, including:

1. Dukoral® (Crucell) consists of a mix of killed whole cell V. cholerae O1 bacteria of both biotypes and serotypes plus 1 mg of cholera toxin B subunit.79,80 2. Shanchol™ (Shanta, Hyderabad, India) contains a mix of killed vibrios of both O1 (both biotypes and serotypes) and O139 V. cholerae.81,82 3. Euvichol® Plus (Eubiologics, Seoul, Korea) contains the identical formulation of vibrios as Shanchol and Euvichol but in a simple, highly practical presentation.83 4. Vaxchora® (PaxVax Bermuda, Ltd., Hamilton, Bermuda [part of PaxVax, Redwood City, CA) live single-dose oral cholera vaccine consists of genetically-engineered V. cholerae O1 strain CVD 103-HgR.3,84,85

A detailed comparison of the salient features of these vaccines is summarized in Table 2.

Table 2. Salient Characteristics of Four Licensed Oral Cholera Vaccines

PxVx0200 Parameter Vaxchora (CVD 103-HgR) Dukoral Shanchol Euvichol Plus of Comparison (CVD 103-HgR) high dose (~ 109 cfu) Components Heat inactivated Heat inactivated Heat inactivated Recombinant Recombinant V. cholerae V. cholerae V. cholerae O1 V. cholerae O1 V. cholerae O1 O1 classical O1 classical classical Inaba classical Inaba classical Inaba Inaba (2.5x1010), Inaba (2.5x1010), (300 Elisa units strain CVD strain CVD classical Ogawa classical Ogawa [EU]), classical 103-HgR with 103-HgR with (2.5x1010), formalin- (2.5x1010), Ogawa (300 deletion of ctxA deletion of ctxA inactivated formalin- EU), formalin- and insertion of a and insertion of a classical Ogawa inactivated inactivated Hg++ resistance Hg++ resistance (2.5x1010), formalin- classical Ogawa classical marker in hlyA marker in hlyA inactivated El Tor (2.5x1010), Ogawa (300 (inactivating (inactivating Inaba (2.5x1010) formalin- EU), formalin- Hemolysin A) (~108 Hemolysin A) (~109 and 1 mg of inactivated El Tor inactivated El Tor colony forming colony forming recombinant Inaba (2.5x1010) Inaba (300 EU) units [cfu]) units [cfu]) cholera toxin B and 1 mg of and formalin- subunit suspended recombinant inactivated in 3 ml of buffer cholera toxin O139 (300 EU) B subunit suspended in 1.5 suspended in 1.5 ml of buffer ml of buffer No. of doses 2 2 2 1 1 Interval between 2 weeks 2 weeks 2 weeks - - doses Well tolerated Yes Yes Yes Yes Yes The high-dose (109 Efficacy or ~ 65% (by cfu) formulation Yes. (79% efficacy effectiveness ~ 50% ~ 65% extrapolation will be used by extrapolation in endemic from Shanchol) in endemic from Orochol E)97 populations populations Efficacy in Yes industrialized Yes No No Yes3,85 (by extrapolation country adults from Vaxchora3,85) VACCINOLOGY IN LATIN AMERICA 10 of Comparison toddlers andpre- Onset of efficacy Immunogenicity immunogenicity immunogenicity

school children Herd immunity in toddlersand in HIV-positive following first delivering the Presentation Parameter Strategy for Vaccines toPrevent Cholera in pregnant pre-school of efficacy Efficacy in responses Boostable Duration immune children Safety & Safety & persons women vaccine dose

of vaccineinaglass The buffer sachetis mixed. Forchildren the 3mlof vaccine emptied intoacup solution shouldbe above, one-halfof sachet withbuffer. Not known.Likely adding the3mlof discarded (leaving added andfurther Liquid suspension water, stirred and the 150mlbuffer an aluminumfoil accompanied by with 150of cool vial containinga (age 2yearsand single doseand 75 ml)before suspension is campaigns Mostly via Dukoral ≥ 21days 3-4 yrs vaccine. Yes Yes Yes Yes Yes Yes 110 106 Liquid suspension the mouthof the single dose.The in olderchildren forceps andthe Likely ≥21days 1.5 mlcontents Yes (lowerthan hand orwitha is removed by cap of thevial transferred to of thevialare containing a in glassvials Not known. Shanchol and adults) campaigns of vaccine Mostly via vaccinee. 5 years Yes Yes Yes Yes Yes 112 111 92 Not known.Likely easily removal tips Liquid suspension plastic tubeswith for direct transfer the mouthof the Euvichol Plus of the1.5mlof from Shanchol from Shanchol from Shanchol from Shanchol from Shanchol from Shanchol directly into to liquid vaccine Extrapolation Extrapolation Extrapolation Extrapolation Extrapolation Extrapolation of vaccinein campaigns Mostly via vaccinee. ≥ 21days Likely data data data (extrapolation from the buffer sachetis lyophilized vaccine the vaccinesachet upnin stirred. suspension put intoacupand vaccine cocktailis (CVD 103-HgR) Yes, butonlyafter sachet containing following primary At least6months at least4months (by extrapolation is addedandthe resultant 100ml Double sachets, The contentsof The contentsof 100 mlof water from Mutacol) Orochol Edata) from Orochol E are then added to reconstitute buffer powder. the lyophilized then ingested. then (extrapolation immunization and theother vaccine. The 8–10 days Travel clinics Vaxchora one sachet containing data) Likely Yes Yes ? ? 107-109 3,96 96 the buffer sachetis lyophilized vaccine the vaccinesachet upnin stirred. suspension put intoacupand vaccine cocktailis (CVD 103-HgR) Yes, butonlyafter sachet containing following primary At least6months at least4months (by extrapolation is addedandthe resultant 100ml Double sachets, The contentsof The contentsof 100 mlof water from Orochol E from Orochol E are then added to reconstitute buffer powder. the lyophilized from Mutacol) then ingested. then (extrapolation (extrapolation immunization and theother vaccine. The 8–10 days Age ≥2years PxVx0200 (~ 10 high dose one sachet campaigns containing Mostly via data data Likely Yes Yes 107-109 ? 9 cfu) 97 ) 3,96 ) Levine and Chen 11

Non-Living Oral Vaccines. Dukoral is the commercial product of a non-living oral cholera vaccine prototype that was tested in U.S. volunteers and then in a randomized controlled field trial in Bangladesh in the 1980s.39,86 The prototype vaccine contained purified B subunit prepared from holotoxin by biochemical separation of the B subunit from the toxic A subunit. The current commercial formulation, Dukoral, contains recombinant B subunit.87 Dukoral has been shown to be well tolerated and protective against cholera in post-licensure evaluations.88,89 The B subunit enhances Dukoral’s anti-bacterial immunity by adding antitoxic immunity that is also effective against enterotoxigenic Escherichia coli producing heat-labile enterotoxin; however, the additive protection of antitoxic immunity is short- lived, lasting only 4-6 months.90,91 Dukoral, administered as two doses 2 weeks apart, is used by European and Canadian travelers for protection against travelers’ diarrhea caused by LT-producing E.coli. Although Dukoral has been pre-qualified by the World Health Organization for procurement by U.N. agencies, heretofore it has been little used for control of endemic or epidemic cholera other than in demonstration projects.

Shanchol demonstrated its ability to diminish the incidence of cholera in highly endemic neighborhoods of Kolkata, India.92 Two doses of Shanchol administered two weeks apart conferred 65% efficacy (95% CI, 52–74%) against cholera overall (all ages combined).92 However, there was a clear hierarchy of protection with young children 1–4 years of age (who suffer the highest incidence of cholera) having the lowest level of efficacy. Over the 5 years of surveillance, the efficacy was 75% in persons ≥ 15 years of age, 68% in children age 5–14 years, and 42% in children 1–4 years of age at the time of enrollment and vaccination.92 The impact of prior immunologic priming was evident during the first year of follow-up when the point estimate of efficacy was only 17% in children age 1–4 years but was 81% in older children age 5–14 years and 66% in individuals age 15 years and above.82 Shanchol was also efficacious in a nested case/control study following a mass vaccination to control seasonal cholera in Guinea;93 this trial also highlighted the complexities of organizing reactive immunization campaigns and the desirability of a single-dose regimen.94 A single-dose of Shanchol was systematically evaluated in a massive randomized placebo-controlled field trial in urban slums in Dhaka, Bangladesh. A single dose gave 63% (95% CI, -39–90%) protection among children 5–14 years of age, 56% (16–77) protection among persons ≥ age 15 years but only 16% (-49–53%) efficacy among children <5 years of age.95 The incidence of cholera in children <5 years (1.75/105 person days) was 8.3-fold higher than among children 5–14 years (0.21/105 person days) and 5.8-fold higher than among persons age ≥ 15 years, presumably indicating the ability of single-dose Shanchol to work well in persons with considerable prior background immunity to cholera but not performing well in immunologically less-primed hosts.95 Heretofore, Shanchol has been the oral cholera vaccine most extensively utilized from the WHO cholera vaccine stockpile.

There are no pre-licensure efficacy or post-licensure effectiveness data yet on Euvichol or Euvichol Plus. They were licensed based on their identity of formulation to Shanchol and clear demonstration of non-inferiority in eliciting seroconversion of serum vibriocidal antibody titers.83 Euvichol and Euvichol Plus received rapid WHO prequalification and will now be able to expand the supply of oral cholera vaccine in the WHO stockpile. In Table 2 it is assumed that Euvichol Plus will provide similar effectiveness as Shanchol.

Vaxchora™ (strain CVD 103-HgR) was licensed by the U.S. FDA in June 2016 and in the U.S.A. and other industrialized country markets will provide a single-dose, rapidly acting (strong protection evident in 8-10 days) oral cholera vaccine for immunologically-naïve persons who must travel on short notice to places of high risk.85 CVD 103-HgR has a deletion of the gene encoding the enzymatically-active A subunit of cholera toxin, while leaving intact the immunogenic B subunit. It also has a Hg++ resistance marker inserted into hlyA, thereby inactivating Hemolysin A expression. In persons from industrialized countries, a single oral dose containing ~108 colony forming units (cfu) of CVD 103-HgR is well tolerated, elicits serum vibriocidal antibody seroconversion in >90% of vaccinees, has only modest excretion (18–25% have positive coprocultures from day 1–4 post-vaccination) and confers 90% efficacy VACCINOLOGY IN LATIN AMERICA Judicious use of these vaccinescandiminish theriskof choleraworldwide. vaccine optionsnowexistto prevent choleradisease.Theglobalsupplyof choleravaccinesinalsoincreasing. industrialized countriesto cholera-endemicandepidemicregions of theworld, severalnewandimproved oral Both for theprevention of diseaseinpopulations incholera-endemiccountriesandfor travelers from Conclusion careful of eating seafood dishesunless theyare cooked toahightemperature. uncooked saladvegetables)should beavoided. Travelers tocholera-endemicareas shouldbeparticularly (boiled orchemicallytreated) water shouldbeconsumed.Foodsthat maybefecally contaminated (e.g., water orfood, enteric precautions shouldbetaken whenlivingortraveling inendemicareas. Onlytreated Safe Water andFood. Prevention andControl serum vibriocidalantibodiesthanoneortwodosesof Shancholusedastheimmunologiccomparator. West Africa,asingledoseof thehigh-doseformulation wassignificantlymore immunogenicinstimulating the manufacturer of Vaxchora toexplore itsutilityfor reactive vaccination. calculated. vaccination undertaken bytheWHOduringacholeraepidemicinMicronesia where 79%vaccineefficacywas Orochol E’sabilitytoprotect populations indevelopingcountrieswaslater showninapost-licensure reactive where efficacycouldnotbedemonstrated butthecholeraburden was greatly diminished for fouryears. that theliveoralvaccineviaindirect protection lowered theoverall incidence inthecommunitytoapoint cholera incidencedropped by>80%inwhat waspreviously ahyperendemic ecology. Oneinterpretation is the vaccinedidnotshowevidenceof significantprotection butshortly after theenrollment andvaccination, in theJakarta trialwhenthecriticalrole of indirect protection wasnotyetappreciated. Jakarta neighborhoodswhere cholerawashyperendemic. Orochol Ewasevaluated inalarge-scale, randomized, placebo-controlled, double-blindedfieldtrialinNorth the needfor thehigherdoseinimpoverisheddevelopingcountrypopulations hasbeenreviewed. to theliveoralvaccine.Thehighernumberof cfuperdoseovercomes thisintestinalbarrier. levels of thepopulation at highestriskof choleraandotherentericinfections, dampenstheimmuneresponse country populations isthat environmental enteropathy, whichishighlyprevalent inthelowsocioeconomic was commercialized for useindevelopingcountries. months after vaccination. either InabaorOgawaserotype andconferred protection againstchallengeassoon8daysandlong6 formulation protected volunteersagainstchallengewith commercialized underthetradenameOrochol CVD 103-HgR wasoriginallymanufactured bythenowdefunct Swiss SerumandVaccine Instituteand identified serconversion of vibriocidalantibodyasastrong correlate of protection. ingestion of asingledose,80%vaccineefficacywas recorded. against challengewithwildtype 12

Vaccines toPrevent Cholera 97 Clinicaltrialshave begunwithahigh-doseformulation of CVD103-HgR (PXVX0200) prepared by Sinceentericfever pathogens are typically acquired viatheingestionof contaminated 96 Aformulation containingone-loghighervaccineorganisms, Orochol E(~10 V. cholerae O1 10 days after vaccination. Upon challenge at 3 months following O110daysafter vaccination. Uponchallengeat 3monthsfollowing ® inmanycountriesandasMutacol 97 Thereason for theone-loghigherdosagefor developing V. cholerae 102 3 ThevolunteerchallengestudieswithVaxchora Randomization wasat thelevelof theindividual O1of eitherElTor orclassicalbiotypeand 85,105 Inonepreliminary studyinMali, 3 ® inCanada.Thisearlier 103,104 Inthisvenue 98-100 Thebiologyof 101

105 9 102 cfu),

Levine and Chen 13

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Vaccines toPrevent Cholera Ciglenecki I, Sakoba K,Luquero FJ etal.Feasibility of MassVaccination CampaignwithOralCholeraVaccines inResponse toan Luquero FJ, Grout L,CigleneckiIetal.Useof Bhattacharya SK,SurD, AliMetal.FiveYear Efficacy ofaBivalentKilledWhole-Cell OralCholera Vaccinein Kolkata, India:ACluster- Peltola H, SiitonenA,Kyrönseppä Hetal. Prevention of Travellers’ Diarrhoea byOralB-Subunit/Whole-Cell CholeraVaccine. Clemens JD, SackDA, HarrisJRet al.Cross-Protection byBSubunit-Whole CellCholeraVaccine AgainstDiarrheaAssociated with Khatib AM, AliM, vonSLetal.Effectiveness of an OralCholera Vaccine inZanzibar:Findings From aMass Vaccination Campaignand Lucas ME,DeenJL,vonSLetal.Effectiveness of MassOral Cholera Vaccination inBeira,Mozambique. Sanchez J, Holmgren J. RecombinantSystem for Overexpression of CholeraToxin BSubunitin Black RE,LevineMM, ClementsML,Young CR,Svennerholm AM, Holmgren J. Protective Efficacy inHumans ofKilledWhole-Vibrio Levine MM, ChenWH, KaperJB, LockM, DanzigL,GurwithM. PaxVax CVD103-HgR Single-Dose LiveOralCholeraVaccine. Levine MM, KaperJB. LiveOralCholeraVaccine: From PrincipletoProduct. Baik YO, ChoiSK,OlvedaRMetal.ARandomized, Non-Inferiority Trial ComparingTwo BivalentKilled,WholeCell,OralCholera Sur D, KanungoS, SahBetal.Efficacy ofalow-Cost, Inactivated Whole-Cell OralCholera Vaccine:Results From 3 Years ofFollow- Kanungo S, LopezAL,AliMetal.VibriocidalAntibodyResponsestoaBivalentKilledWhole-Cell OralCholeraVaccine inaPhaseIII Taylor DN, Cardenas V, SanchezJLetal.Two Year Study of theProtective Efficacy oftheoralWholeCellPlusRecombinantBSubunit Sanchez JL,Vasquez B, BegueREetal.Protective Efficacy ofOralWhole-Cell/Recombinant-B-Subunit Cholera VaccineinPeruvian Alam AN, AlamNH, AhmedT, SackDA. RandomisedDoubleBlindTrial of SingleDoseDoxycycline for Treating CholerainAdults. Nair GB, ShimadaT, Kurazono Hetal.Characterization of Phenotypic,Serological, andToxigenic Traits of Khan WA, SahaD, RahmanA,SalamMA,BogaertsJ, BennishML.Comparisonof Single-Dose Azithromycin and12-Dose, 3-day Khan WA, BennishML,SeasCetal.RandomisedControlled Comparisonof Single-Dose Ciprofloxacin andDoxycycline for Cholera Saha D, KarimMM, KhanWA, AhmedS, SalamMA,BennishML.Single-Dose Azithromycin for theTreatment of CholerainAdults. Saha D, KhanWA, KarimMM, ChowdhuryHR,SalamMA,BennishML.Single-Dose Ciprofloxacin Versus 12-Dose Erythromycin for Gotuzzo E,SeasC, Echevarria J, CarrilloC, MostorinoR,RuizR.Ciprofloxacin for the Treatment of Cholera:aRandomized, Double- J ClinMicrobiol Vibrio Cholerae 2006;354:2452-2462. 2017;16:197-213. Proc Natl AcadSci Lancet Plos Me PLoS ONE 1994;32:2775-2779. 1994;344:1273-1276. 01or0139. d 2013;10:e1001512. Lancet Infect Dis 2014;9:e96499. U SA1989;86:481-485. J Infect Dis PLoS NeglTrop Dis Lancet 2012;12:837-844. 1996;348:296-300. Escherichia coli 2000;181:1667-1673. Vaccine Vibrio Cholerae Lancet LancetInfect Dis 2011;5:e1289. 2015;33:6360-6365. 2005;366:1085-1093. : Resultsof aLarge-Scale FieldTrial. Vaccine inanOutbreak inGuinea. 2013;13:1050-1056. Clin Infect Dis Infect Immun Lancet 2002;360:1722-1727. BullInstPasteu 1987;55:1116-1120. 1995;20:1485-1490. r 1995;93:243-253. J Infect Dis VibrioCholerae N EnglJMed N EnglJMed 1988;158:372-377. Vibrio Cholerae 2014;370:2111-2120. asaBasisfor Vaccine 2005;352:757- O139 Lancet Expert BMJ N

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